User`s Manual
Contents
1. a division of Harvard Bioscience Inc ben AgilePulse MAX Large Volume Transfection system USER S MANUAL www btxonline com Publication 015 101446 Rev 1 0 AgilePulse MAX Large Volume Transfection System RESEARCH ONLY BTX Harvard Apparatus 84 October Hill Rd Holliston MA 01746 USA phone 1 508 893 8999 fax 1 800 429 5732 www btxonline com BIX Warranty Harvard Apparatus warranties the AgilePulse MAX Large Volume Transfection System for a period of two years from the date of purchase At its option BTX Harvard Apparatus will repair or replace the unit if it is found to be defective as to workmanship or materials This warranty does not extend to any instrumentation which has been a subjected to misuse neglect accident or abuse b repaired or altered by anyone other than BTX HARVARD APPARATUS without BTX HARVARD APPARATUS express and prior approval c used in violation of instructions furnished by BTX HARVARD APPARATUS This warranty extends only to the original customer purchaser IN NO EVENT SHALL BTX HARVARD APPARATUS BE LIABLE FOR INCIDENTAL OR CONSEQUENTIAL DAMAGES Some states do not allow exclusion or limitation of incidental or consequential damages so the above limitation or exclusion may not apply to you THERE ARE NO IMPLIED WARRANTIES OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR USE OR OF ANY OTHER NATURE Some states do not allow this limitation on an implied2. Membrane Electrode 1 Electrode 2 Figure 3 2 TRANSMEMBRANE VOLTAGE 3 3 PulseAgile Electroporation PulseAgile electroporation differs from standard electroporation in that it allows control of pulse parameters from pulse to pulse within a single delivery sequence This means that values for pulse voltage pulse duration interval between pulses and number of identical pulses in a subset can be programmed to change during a series of pulses In practice PulseAgile is usually used to deliver high electric field pulses of short duration followed by lower electric field pulses of longer duration This method increases electroporation efficiency in numerous applications and is covered by U S patents 6 010 613 and 6 078 490 Physical principles of importance to electroporation are 1 Applied pulsed electric fields of optimal magnitude and duration induce a transient state of permeability in cells immersed in that electric field Neumann 1972 2 An applied electric field can move charged particles in an ionic medium by electrophoresis Moyer 1936 3 An electrical current is required to maintain an electric field in an ionic medium Ohm s Law 4 Heat production by an electrical current in an ionic medium is proportional to the square of the current JJoule s First Law These principles can be used to optimize electroporation All four of these physical processes are active during the entire time that electric fiel
3. 1 Emergency Flash memory storage failure 2 Waveform protocol data integrity verification error 3 Waveform protocol mode not recognized 4 Protocol voltage below minimum specification 5 Protocol voltage above maximum specification 6 Protocol pulse width below minimum specification 7 Protocol pulse width above maximum specification 8 Protocol pulse interval below minimum specification 9 Protocol pulse interval above maximum specification 10 Protocol pulse number below minimum specification 11 Protocol pulse number above maximum specification 12 Protocol has a zero pulse group in between two non zero pulse groups 13 Protocol pulse settings exceed maximum duty cycle 14 Low voltage power supply failure Hard system turn off 15 High volt power supply voltage above maximum voltage 16 High volt power supply maximum current draw 17 Pulse voltage monitor detected off zero voltage while no pulse was active 18 Pulse voltage monitor below maximum droop specification 19 Pulse voltage monitor above maximum overshoot specification 20 Pulse current monitor below minimum specification 21 Pulse current monitor above maximum specification 22 Independent time audit system failed to respond to query 23 Independent time audit system detected less pulses than expected 24 Independent time audit system detected more pulses than expected 25 Independent time audit system exceeded maximum measurable pulse number 26 Independent time audit system detected
4. 1200 oO 0 00 0 0 050 0 000 0 200 0 000 Pre pulse Estimated Load 5000 Ohms Average Monitored Pulse Load 0O Ohms Figure 5 4 Pulse Delivery Screen Post Pulse User s Manual 5 1 Waveform Generator cont d 5 1 4 User Account Maintenance The user account maintenance screen is accessed from the Welcome screen Touch SETUP Touch USER User accounts and privileges are managed from this screen While standard users will only see the Active User section and thus will only be able to change their password the system administrator has access to account properties for all standard users As an administrator a new user account can be adding by pressing the Add New User button Once the account Is created the new user is assigned read only access to all of the pulse protocol waveforms on the system The administrator may then select individual protocols and provide more access by giving both read and write privileges or they may restrict access to a protocol altogether by giving the user no access at all When a user Is not given any protocol access they will not be able to see or select it from the Protocol screen when they login under their own user name When protocols are added through the Protocol screen after a standard user account has already been created all user accounts in the system will receive read only access to the new protocol The administrator also has the ability to change a user s password This is
5. 125 000 125 000 125 000 125 000 10 16 40 PM GMT 125 075 125 083 125 081 125 082 125 081 125 081 125 082 125 080 125 083 52 879 User s Manual BIX a division of Harvard Bioscience Inc 24 Gp AgilePulse MAX System Operation 5 1 Waveform Generator cont d 5 1 7 Electroporation Protocol Delivery Logs cont d 5 1 7 2 CSV Data Detailed pulse information is exported from raw data and formatted to a standard comma separated file primarily meant to be imported into Microsoft Excel for further user manipulation As an example in Figure 5 9 a chart was made to displays the monitored amplitude samples of three 100 microsecond pulses run at 5 kHz In this case each pulse contains eight amplitude samples Time vs Amplitude 500 450 400 350 300 250 200 150 100 50 Amplitude volts 0 1 0 0 1 0 2 0 3 0 4 0 5 0 6 Time ms Figure 5 9 Sample Amplitude Analysis E AgilePulse MAX Large Volume Transfection System User s Manual ae toll free 800 272 2775 508 893 8999 www btxonline com 25 AgilePulse MAX System Operation 5 1 Waveform Generator cont d 5 1 8 Delivery Completion Return Codes Several return codes are available following system shutdown describing how it took place Return Code Description 0 Successful completion Normal Operation
6. 2010 3rd Edition Emissions Immunity EN 61326 1 2013 FR47 FCC Part I5 Sub Part B 2011 EN 61000 4 2 1995 A1 1998 A2 2001 EN 61000 4 3 2002 EN 61000 4 4 2004 EN 61000 4 5 1995 A1 2001 EN 61000 4 6 2007 EN 61000 4 11 2004 EN 61000 3 2 2000 EN 61000 3 3 1995 A1 2001 CISPR11 2003 A1 2004 Group I Class A EMC and Safety compliance were evaluated by Intertek ETL Semko Reference test report file numbers 100587731 BOX 001 003 004 I the undersigned hereby declare that the equipment specified above conforms to the above Directive s and Standard s Place United States of America Date April 14 2015 th Signature C 4 Mark Davis Full Name k Pavara Director Global MPE Apparatus Position
7. AgilePulse MAX Large Volume Transfection System User s Manual ae toll free 800 272 2775 508 893 8999 e www btxonline com 1 Table of Contents SUBJECT 1 Introduction 2 Items of Particular Interest 3 AgilePulse MAX Tutorial 3 1 Applications7 3 2 DNA RNA Delivery Into Cells Using Electroporation 3 2 1 General Electroporation Discussion 3 3 PulseAgile Electroporation 3 3 1 Induction of a State of Permeability 3 3 2 Movement of DNA or RNA Across Cell Membranes 3 3 3 Minimizing Heat Production with Waveform 3 3 4 Putting the AgilePulse MAX Concepts Together 3 4 Importance of Medium Conductivity in Electroporation 3 5 Cytoporation Medium Properties 3 5 1 Conductivity 3 5 2 Media Formulas 3 5 3 Other Important Cytoporation Medium Properties 3 6 References 4 AgilePulse MAX System Components and Set up 4 1 Introduction 4 2 The AgilePulse MAX Waveform Generator 4 2 1 Front Panel 4 2 2 Back Panel 4 2 3 Large Volume Chamber and Base 4 2 4 Cytoporation Medium 4 3 Setting Up the System 4 3 1 Initial System Test 4 3 2 Touch Screen Cleaning 5 AgilePulse MAX System Operation 5 1 Waveform Generator 5 1 1 Login Screen User Identification Input 5 1 2 Welcome Screen Sample Identification Input 5 1 3 Pulse Delivery Screen Functions and Operation 5 1 4 User Account Maintenance 5 1 5 Protocol Setup 5 1 6 Log File Maintenance 5 1 7 Electroporation Protocol Delivery Logs 5 1 7 1 Text Log Oe bat a2 CSV LOG 5 1 8 D
8. Waveform Generator Subsystem contains a high voltage power supply and was designed with safety features to protect the user and the equipment If used properly the AgilePulse MAX Waveform Generator is a safe and reliable instrument Front and Back Panel Symbols ATA O Caution Risk of Operating Caution On Fuse AC Mains Electric Shock Instructions The AgilePulse MAX system is intended for laboratory use only and can be used in research and development applications The system was designed to meet the standards for electromagnetic compatibility EMC and safety requirements intended for laboratory equipment applications This product should not be used in the presence of a flammable atmosphere such as an anesthetic mixture with air oxygen or nitrous oxide The following material is contained in this User Manual CHAPTER 2 Items of Particular Interest CHAPTER 3 AgilePulse MAX Tutorial CHAPTER 4 AgilePulse MAX System Components and Set up CHAPTER 5 AgilePulse MAX System Operation CHAPTER 6 Customer Service T AgilePulse MAX Large Volume Transfection System User s Manual ae toll free 800 272 2775 508 893 8999 e www btxonline com 5 Ep items of Particular Interest The AgilePulse MAX Large Volume Transfection System is the most sophisticated system available The system is available from BTX Harvard Apparatus Inc The following are important 1 7 8 The system shall o
9. following successful user login that contains fields that should be completed before the electroporation process is initiated Information is entered into each box by a virtual keyboard Entering data into each field is optional Fields may be left blank before proceeding to the next screen To change pulse parameters touch SETUP before advancing to pulse initiation Pre programmed protocols are selected from a drop down menu on the PROTOCOLS screen see Section 5 1 5 Information entered into the data fields will be retained when returning from SETUP The data fields are cleared upon completion of pulse delivery When all of the fields are filled in and the desired protocol has been selected pulse delivery Is initiated by touching NEXT to advance to the Pulse Delivery Screen Cell Line a Welcome to Transfectant Chamber Protocol Operator Plasmid A MDO81101 1 Agile Pulse MAX Default admin Log Out Figure 5 2 Welcome Screen 5 1 3 Pulse Delivery Screen Functions and Operation The Pulse Delivery Screen is used to control the delivery of electroporation pulses to the sample The Pulse Delivery Screen also provides visual feedback before during and after pulse delivery Procedure for Pulse Delivery 1 Fill a Large Volume Electroporation Chamber with 2 to 6 mLs cell suspension plus polynucleotide such as plasmid DNA Insert the Chamber in the Chamber Holder Do not insert fingers or other obje
10. ideal for situations in which the user forgets his her password and thus needs the password to be reset in order to login again AgilePulse MAX System Operation Active User User Name admin oe Password e Accounts Password Protocol Access ior Geos vom Figure 5 5 User Account Screen IMPORTANT The changes made on this screen will only be permanently saved when the HOME button is pressed to go back to the Welcome Screen If modifications are made and system power is turned off Caution while still viewing the screen all changes will be lost AgilePulse MAX Large Volume Transfection System User s Manual toll free 800 272 2775 508 893 8999 www btxonline com 20 5 1 Waveform Generator cont d 5 1 5 Protocol Setup Pulse protocols can be viewed and or modified on the Protocol Screen which is accessed from the Welcome Screen Touch SETUP Touch PROTOCOL Parameters for up to three pulse groups are set by selecting the appropriate box and scrolling through the available values Subsequent groups are activated by selecting more than one pulse in the number field and are conversely deactivated by selecting zero pulses in the number field The adjusted protocol is saved to internal memory when the HOME button is touched Only the system administrator has access to the Add New and Remove buttons which create new protocols and delete existing protocols
11. respectively When a new protocol is created all user accounts in the system receive read only access to it The administrator can customize user access privileges for each protocol by navigating to the User Maintenance screen see Section 5 1 4 Default Add New Remove Group 1 Group2 Group 3 1200 too po 100 _ ae 0 050 0 0 MS Protocol Pulse Amplitude Duration Interval 0 2 4 lo ie m B E Number 1 0 Sound M Enable Multi Cycle Enable r Figure 5 6 Protocol Screen IMPORTANT The changes made on this screen will only be permanently saved when the HOME button is pressed to go back to the Welcome Screen If modifications are made and system power is turned off while still viewing the screen all changes will be lost Caution AgilePulse MAX System Operation AgilePulse MAX Large Volume Transfection System toll free 800 272 2775 508 893 8999 e www btxonline com 21 There are limitations on the range of parameters and interaction between various parameters The following rules must be observed Pulse Amplitude 100 to 1200 10 Volt Steps Pulse Width 50 us to Volts Range 1000 us 100 to 300 10 Volt Steps Pulse Width 1 ms to Volts Range 10 ms Pulse Drop Less than 2 of power supply voltage at 100 ohms Less than 8 of power supply voltage at 10 ohms Pulse Widths in ms 0 05 0 10 0 30 0 40 0 50 1 2 3 4 5 10 Pulse Width Droop lt 7 5 Puls
12. typical concentrations of polynucleotides that are used for electroporation e DNA 10 to 100 g mL e mRNA 10 to 100 g mL e siRNA 100 nM to 1 uM e Oligonucleotides 100 nM to 20 uM 5 2 2 Preparing Cell Suspensions for Large Volume Transfection The following is a standard procedure for preparing cells for large volume electroporation Cell washes may be carried out at 4 C However it is recommended that electroporation in Cytoporation media be carried out at room temperature Harvest cells from several T150 flasks roller bottles or fermenter Centrifuge cells 300 X g for 10 minutes Pour off supernatant resuspend cells and add 20 mL Cytoporation Medium T Centrifuge cells 300 X g for 10 minutes Pour off supernatant resuspend cells and add 20 mL Cytoporation Medium T Centrifuge cells 300 X g for 10 minutes AMAAN R Resuspend cells at 10 to 100 million cells ml in Cytoporation Medium T or T 4 8 Add polynucleotide DNA or RNA to desired concentration Efficient transfection can be achieved with cell densities ranging from 10 million cells mL to 100 million cells mL It is important to remember that at high cell densities leakage of ions from the cells during electroporation can contribute to significant increases in conductivity during pulse delivery 5 2 3 Filling the Chamber The Large Volume Electroporation Chamber can be filled through one of the luer lock ports It is recommended that a sterile syringe be used f
13. warranty so the above limitation may not apply to you Without limiting the generality of the foregoing BTX HARVARD APPARATUS shall not be liable for any claims of any kind whatsoever as to the equipment delivered or for non delivery of equipment and whether or not based on negligence Warranty is void if the AgilePulse MAX instrument is changed in any way from its original factory design or if repairs are attempted without written authorization by BTX HARVARD APPARATUS Warranty is void if parts connections or electrodes not manufactured by BTX HARVARD APPARATUS are used with the AgilePulse MAX instrument If a defect arises within the warranty period promptly contact BTX Harvard Apparatus 84 October Hill Road Building 7 Holliston Massachusetts USA 01746 1388 using our toll free number 1 800 272 2775 US Only or 508 893 8999 E mail techsupport btx harvardapparatus com Goods will not be accepted for return unless an RMA Returned Materials Authorization number has been issued by our customer service department The customer Is responsible for shipping charges Please allow a reasonable period of time for completion of repairs replacement and return If the unit is replaced the replacement unit is covered only for the remainder of the original warranty period dating from the purchase of the original device This warranty gives you specific rights and you may also have other rights which vary from state to state o
14. 000 0 4 2 1000 0 8 2 DBS FRhL 2 mRNA 0 24 4500 0 1 2 1250 0 4 4 Neuro 2a Plasmid 50 1260 0 4 2 630 1 2 K 562 Plasmid 25 2500 0 1 6 VERO Plasmid 50 2000 0 2 2 1000 1 4 5 2 5 Cell Handling After Electroporation Cytoporation Media are non toxic to cells but do not have nutrients to support cell growth Therefore cells should be kept in Cytoporation Medium for a limited time It is recommended that cells be suspended in Cytoporation Media for a maximum of one hour prior to electroporation It is recommended that cells be returned to culture medium soon after electroporation However transfection efficiency is often enhanced by allowing the cells to rest for 1 2 minutes before adding culture medium The cells can be either washed in culture medium to completely remove the Cytoporation Medium or the cells can be diluted in culture medium without removing the Cytoporation Medium It is recommended that at least five parts culture medium be added for every one part Cytoporation Medium T AgilePulse MAX Large Volume Transfection System User s Manual ae toll free 800 272 2775 508 893 8999 www btxonline com 28 6 Customer Service 6 1 Limited Warranty The terms of the warranty are covered in the BTX warranty 6 2 Customer Service If the user believes that there is a defect in the BTX product the customer should contact BTX Technical Support at 800 272 2775 or email techsupport btx h
15. J C 1989 The number of molecules taken up by electroporated cells quantitative determination FEBS Lett 256 4 10 Chang D C Chassy B M Saunders J A and Sowers A E eds 1992 Guide to Electroporation and Electrofusion Academic press San Diego 581 pp Dimitrov D S and Sowers A E 1990 Membrane electroporation fast molecular exchange by electroosmosis Biochimica et Biophysica Acta 1022 381 392 Disalvo E A and S A Simon eds 1995 Permeability and Stability of Lipid Bilayers CRC Press Boca Raton p 105 121 Djuzenova C S Zimmermann U Frank H Sukhorukov V L Richter E amp Fuhr G 1996 Effect of medium conductivity and composition on the uptake of propidium iodide into electropermeabilized myeloma cells Biochim Biophys Acta 1284 143 152 Golzio M Teissie J Rols MP Direct visualization at the single cell level of electrically mediated gene delivery 2002 Proc Natl Acad Sci U S A Feb 5 99 3 1292 7 Klenchin VA Sukharev SI Serov SM Chernomordik LV Chizmadzhev YA Electrically induced DNA uptake by cells is a fast process involving DNA electrophoresis 1991 Biophys J 60 804 811 Moyer LS A suggested standard method for the investigation of electrophoresis 1936 J Bacteriology 31 5 531 546 Neumann E Kakorin S amp Toensing K 1999 Fundamentals of electroporative delivery of drugs and genes Bioelectrochem Bioenerg 48 3 16 Neumann E
16. MD081101 1 Process Completion Return Code 0 Success Pulse VSet VMon IMon RMon AgilePulse MAX System Operation Field Description Pulse Pulse number VSet Voltage programmed to high voltage power supply VMon_ Voltage monitored during pulse IMon Current monitored during pulse RMon_ Equivalent load resistance calculated from voltage and current monitors R V I DurSet Pulse duration programmed to waveform generator DurMon Pulse duration monitored by independent time auditor system IntSet Pulse interval programmed to waveform generator IntMon Pulse interval monitored by independent time auditor system 2 12 2007 DurSet DurMon 10 16 22 PM GMT IntSet IntMon 450 450 110 110 110 110 110 110 110 1 2 3 4 5 6 7 8 9 1 110 Load Reading Date Stop Time Elapsed 449 449 109 108 108 108 108 109 109 109 204 2 12 2007 00 00 18 2 316 2 316 0731 Ose Toa 0 743 0 747 0 736 0 736 0 732 0 758 2 16 40 PM 194 194 145 147 146 145 147 147 148 143 2 12 2007 0 050 0 050 10 000 10 000 10 000 10 000 10 000 10 000 10 000 10 000 Figure 5 8 Text Log Screen 0 050 0 050 LO 013 10013 10 013 10 013 10 013 10 013 10 013 10 012 AgilePulse MAX Large Volume Transfection System toll free 800 272 2775 508 893 8999 e www btxonline com 125 000 125 000 125 000 125 000 125 000 125 000
17. Rosenheck K Permeability changes induced by electric impulses in vesicular membranes 1972 J Membr Biol 10 279 290 Neumann E Toensing K Kakorin S Budde P amp Frey J 1998 Mechanism of electroporative dye uptake by mouse B cells Biophys J 74 98 108 Neuman E Sowers A E and Jordan C A eds 1989 Electroporation and Electrofusion in Cell Biology Plenum Press New York 581 pp Nickoloff Jac A ed 1995 Plant Cell Electroporation and Electrofusion Protocols Methods in Molecular Biology Volume 55 Humana Press Totowa New Jersey Hayt Engineering Electro Magnetics 1974 McGraw Hill AgilePulse MAX Large Volume Transfection System User s Manual toll free 800 272 2775 508 893 8999 e www btxonline com 13 4 1 Introduction The AgilePulse MAX system will transfect a large volume of cells in a single electric field application The system is designed to be easy to set up and to operate in a research environment The AgilePulse MAX System is composed of five subsystems e Touch screen controlled Waveform Generator e 6 ml large volume chambers Cable Base and Chamber Electrode e Safety Stand Used for Cuvette Electroporation e Cytoporation Medium e User Manual 4 2 The AgilePulse MAX Waveform Generator The Waveform Generator is a computer with high voltage pulsing circuits Figure 4 1 The computer runs mobile Windows 6 0 and is operated via a touch
18. Tutorial 3 5 Cytoporation Medium Properties 3 5 1 Conductivity The conductivity of most common electroporation buffers range from 10 to 20 mS cm these conductivities are too high for electroporation of large volumes Cytoporation media was developed to address this problem Although very low conductivity is desired for large volumes experimental data indicate that some cell types require a moderately conductive environment to achieve optimal transfection efficiency The trade off between maximum volume and optimal efficiency must be considered when choosing a medium for large volume electroporation 3 5 2 Media Formulas There are a number of Cytoporation formulas currently available Cytoporation Medium T at 0 08 mS cm and Cytoporation Medium T 4 at 3 45 mS cm These media are produced under strict standards and are listed with the FDA under Master File Number MAF 1508 Some cell types can be sensitive to the conductivity of the electroporation buffer Therefore optimization Is usually required to determine the most effective formulation for a given cell type Another point to consider is that ions within the cells will leak out when the cell membranes become porated This will contribute to an increase in conductivity of the extracellular solution during the course of electroporation This effect is especially important to consider when working with high concentrations of cells i e 100 million cells mL 3 5 3 Other Important Cy
19. arvardapparatus com If the system needs to be returned to BTX please contact Technical Support for a Return Material Authorization RMA number 6 3 Parts and Accesories Order Description 47 2000 AgilePulse MAX System Includes AgilePulse Max pulse generator cuvette safety stand electroporation stand 2x 6 ml chambers with 6 mm gap manual and CD with training videos 47 0201 AgilePulse Max Generator ONLY 47 0202 Electroporation Stand for 6 ml chambers 47 0203 Saftey stand for cuvettes 47 0010 6 ml chambers 6 mm gap each 47 0002 Cytoporation Media T AgilePulse MAX Large Volume Transfection System User s Manual toll free 800 272 2775 508 893 8999 e www btxonline com 29 Declaration of Conformity Manufacturer Harvard Apparatus Inc 84 October Hill Road Holliston Massachusetts 01746 1388 U S A Phone 508 893 8999 We herewith declare that the following product Product Name BTX AgilePulse ID BTX AgilePulse IM BTX AgilePulse MAX Model No Catalog 47 04xx Catalog 47 05xx Catalog 47 02xx To which this declaration relates is in conformity with the applicable EC Directives harmonized standards and other normative requirements Application of Council Directive s 2006 95 EC Low Voltage Directive 2004 108 EC Electromagnetic Compatibility Directive 2011 65 EU RoHS Compliant Standard s to which conformity is declared Safety IEC 61010 1
20. cts in the Chamber Holder Touch the Load button The system will automatically deliver a short low voltage pulse to estimate the load resistance If the resistance Is too low the pulse will not be delivered If the resistance is within acceptable limits the Ready button will turn yellow Touch the Ready button The system will charge the high voltage capacitors to the set voltage When the system is charged the Start button will turn yellow 5 Touch the Start button The Figure 5 3 Pulse Delivery AgilePulse MAX Large Volume Transfection System toll free 800 272 2775 508 893 8999 e www btxonline com 19 Screen Pre Pulse system will deliver the set pulse protocol When delivery is complete the Done button will turn green and the pulse log will be displayed in the text box Touch the Done button to return to the Welcome Screen The identification fields will be reset Repeat the procedure for each pulse delivery Cell Line CHO System volts Plasmid A Est Load gt ohms MD081101 1 Default Transfectant Chamber Protocol Operator admin Figure 5 3 Pulse Delivery Screen Pre Pulse Cell Line System aal volts Plasmid A Mon Load j ohms MD081101 1 Default Transfectant Chamber Protocol Operator admin Start 3 15 2012 1 15 44 PM 3 x 2012 Process Completion Return Code or 32 2 15 44 PM GMT ElectrodeInter lockOpen Pulse VSet PYMon PIMon RMon DurSet DurMon IntSet IntMon
21. ds are applied to the area around cells However they vary in importance at different times during electroporation User s Manual 3 3 1 Induction of a State of Permeability Applying an electric field across a cell causes a redistribution of internal ions because cell membranes act as insulators lons move according to their charge within the electric field but remain trapped within the cell thus accumulating at the poles in line with the electric field This polar accumulation of ions within the cell creates an electrical potential across the cell membrane When the transmembrane voltage exceeds a threshold of approximately one volt conformational changes within the membrane induce a state of permeability The potential across a cell membrane is not equal along the entire surface of the cell It is highest when the surface of the cell membrane is perpendicular to the electric field in line with the electric field and lowest when the surface of the cell membrane is parallel or tangential to the electric field out of line with the electric field The variation of transmembrane potential in an electric field as described above is related to the cosine of with the greatest transmembrane potential at the poles of the cells in line with the electric field and the least transmembrane potential 90 degrees from the poles See Figure 3 2 Increasing the electric field from one value above threshold to a higher value above threshold will
22. e Intervals in ms 0 2 0 3 0 4 0 5 1 2 3 4 5 10 20 30 40 50 100 200 300 400 500 1000 Inter group Pulse interval must be 2X pulse width with a 0 2 ms minimum Intra group Pulse interval must be 2X pulse width with a 0 2 ms minimum Time between end of pulse N and beginning of pulse N 1 must be gt 0 04 In Vn 1 Vn Number of Pulses 1 to 10 in steps of 1 Number of Groups 1 2 o0r3 User s Manual 5 1 Waveform Generator cont d 5 1 6 Log File Maintenance The Log file maintenance screen Is accessed from the Welcome screen Touch SETUP Touch LOGS All previous transfection run logs can be retrieved and saved onto a USB Key or deleted from internal memory The interface consists of a series of chronological sub folders from which the user is allowed to double tap on the touch screen to descend through the hierarchy The standard naming sequence is Year Month Day followed by special folders with the time of day These time folder icons show a hand underneath meaning they contain all files generated from a single run The folders allow the user to return to the previous level of hierarchy Special time folders display basic information on the right hand side of the screen when selected Identification numbers time of day and status completion indicators help the user find the correct transfection run to download When selected by a single tap on the touch screen icon any
23. e beginning of electroporation to induce permeability in a large area of the cells followed by delivery of a series of lower electric field pulses to induce maximum movement of DNA with minimal heating User s Manual AgilePulse MAX Tutorial 3 4 Importance of Medium Conductivity in Electroporation When using standard cuvettes in electroporation medium conductivity is generally not a critical issue However for large volumes medium conductivity quickly becomes critical As the volume of cell suspension gets larger the resistance of the bulk solution gets smaller This relationship can be represented mathematically as follows R r 2 _ ohms a area R is the resistance in ohms of the solution in the chamber gap isthe spacing between two parallel electrodes area isthe active area of the electrodes that are in contact with the cell suspension the areas of the two electrodes are identical r is the resistivity of cell suspension in the chamber with units ohm cm Engineers and physicists generally use resistivity biologists often use conductivity P 1 Conductivity s Siemens cm resistivity often converted to milliSiemens cm Since It is often difficult to make a direct measurement of the active area of the electrodes in a cuvette or electroporation chamber a more practical equation is needed The volume of cell suspension added to a cuvette can be represented as follows 2 R lt oms volu
24. ed on the top of the chamber is fitted with an air filter and acts as a vent during electroportion The cells Cytoporation medium and material to be transfected are mixed and place in the chamber via the lower luer lock connector close the valve and remove syringe The chamber is then placed into the base The chamber can only be inserted into the base in one direction The end with the plastic protrusion must face the Base to be inserted properly See Figure 4 3 The chamber can be used with a minimum of 2 mL anda maximum of 6 mL The chamber should be used one time unless a validated cleaning procedure Is established documented and allowed by the protocol Figure 4 3 Chamber and Base User s Manual a division of Harvard Bioscience Inc BIX 15 4 2 The AgilePulse MAX Waveform Generator con td Figure 4 4 Cytoporation Medium 4 2 4 Cytoporation Medium BTX Harvard Apparatus provides a low conductivity media that are especially formulated for large volume electroporation There are currently three formulas available with different conductivities Parameter Specification Sterility Sterile filtered pH 7 2 Conductivity 0 08 mS cm Medium T 3 45 mS cm Medium T 4 Endotoxin lt 0 2 IU ml Osmolarity 270 290 milliosmoles Storage 4 to 8 C After Opening Packaging 500 ml bottle See Chapter 3 for more details about conductivity and large volume t
25. eld is applied for twice a given time 3 3 3 Minimizing Heat Production with Waveform Joule s first law states that ohmic or Joule heating is proportional to electrical resistance times the square of the current Joule heating is a common cause of cell death during electroporation SO excessive increases in temperature due to this effect should be avoided Following enough poration of the cell membrane to physically allow transfectant entry a subsequent reduction in electric field will continue to move the transfectant by way of electrophoresis while generating much less heat For example a half decrease in electric field will reduce heat generated four fold 3 3 4 Putting the AgilePulse Concepts Together A summary of the proportional relationships of physical factors during electroporation are 1 The area of a cell permeabilized by electroporation is positively correlated to the magnitude of an applied electric field less than linearly because of the cosine in the formula 2 The movement of a charged molecule such as DNA in a solution in an electric field is linearly related to the total applied charge 3 Heat produced during electroporation is proportional to the square of the applied current or of the applied voltage if the electrical resistance is constant Given these relationships maximum cell permeability and maximum DNA delivery can be achieved by delivering a series of brief high electric field pulses at th
26. elivery Completion Return Codes 5 2 Transfection Process 5 2 1 Preparing the Polynucleotide 5 2 2 Preparing Cell Suspensions for Large Volume Transfection 5 2 3 Filling the Chamber 5 2 4 Electroporation 5 2 5 Cell Handling After Electroporation 6 Customer Service 6 1 Limited Warranty 6 2 Customer Service 6 3 parts and accessories Declaration of Conformity T E E AgilePulse MAX Large Volume Transfection System LO LO WO WO WO 00 10 11 User s Manual toll free 800 272 2775 508 893 8999 e www btxonline com Table of Contents LIST OF FIGURES PAGE 3 1 Electroporation 7 3 2 Transmembrane Voltage 8 4 1 Waveform Generator 14 4 2 Waveform Generator Front Panel 15 4 3 Chamber and Base 15 4 4 Cytoporation Medium 16 4 5 Start Up Screen Display 16 5 1 Login Screen 18 5 2 Welcome Screen 19 5 3 Pulse Delivery Screen Pre Pulse 19 5 4 Pulse Delivery Screen Post Pulse 19 5 5 User Account Screen 20 5 6 Protocol Screen 21 5 7 Log File Maintenance Screen 22 5 8 Text Log Screen 24 5 9 Sample Amplitude Analysis 25 T AgilePulse MAX Large Volume Transfection System User s Manual ae toll free 800 272 2775 508 893 8999 e www btxonline com 3 Caution Notice This instrument contains a high voltage power supply adjustable to 1 000 volts High voltage power supplies present a serious risk of personal injury if not used in accordance with design and or use specifications if used in applications on products for
27. excessive pulse duration variance 27 Independent time audit system detected excessive pulse interval variance 28 Independent time audit system data integrity verification failure 29 Reset button on front panel triggered 30 Control system watchdog timeout 251 High voltage timeout 252 Load estimation timeout 253 Delivery process was terminated prematurely 254 Control system experienced a system reset of unknown source 255 Load estimate outside of allowable range AgilePulse MAX Large Volume Transfection System User s Manual toll free 800 272 2775 508 893 8999 e www btxonline com BIX a division of Harvard Bioscience Inc 26 AgilePulse MAX System Operation 5 2 Transfection Process The transfection process consists of the following steps 1 Preparing the polynucleotide 2 Preparing the cell suspension 3 Filling the chamber 4 Electroporation 5 Handling after electroporation 5 2 1 Preparing the Polynucleotide Transfection efficiency is often directly related to polynucleotide concentration However the optimum concentration may vary depending on the material to be transfected Some optimization will be required for each combination of polynucleotide and cell type It is recommended that the polynucleotide by be prepared in nuclease free water rather than TE or other tonic buffers in order to maintain the low conductivity environment necessary for large volume transfection The following are
28. factors Pores are induced when the product of the pulse amplitude Field Removed Membrane Seals and the pulse duration is above a lower limit threshold Normal cell to cell biological variability can cause some cells to be more Figure 3 1 Electroporation sensitive to electroporation than other cells Although other factors are Involved the lower limit threshold for a particular cell type is largely dependent on the reciprocal of cell size T AgilePulse MAX Large Volume Transfection System User s Manual ae toll free 800 272 2775 508 893 8999 e www btxonline com 7 3 2 DNA RNA Delivery into Cells Using Electroporation cont d 3 2 1 General Electroporation Discussion cont d As charge accumulates at the membrane the voltage across the membrane increases Neumann et al 1989 described the equation that relates the transmembrane voltage TMV to electric field intensity TMV Y Er cosa where E electric field intensity in volts cm r the cell radius in cm Ql angle off the center line Pores in the membrane will begin to form as the voltage increases from its quiescent value of a few tenths of a volt to more than 0 5 volts To produce a TMV of 1 volt across the membrane of a cell with 7 um radius the required electric field intensity is 1 E A 7 104 950 volts cm The number of pores and effective pore diameter increase as the product of pulse amplitude and duration increase At t
29. gured with only the admin account available By default no password Is defined so the user can enter the system with parameters matching that of Figure 5 1 on first login The password may then be changed from the User account screen in Setup section As default the most recently logged in user will be displayed in the User Name field on startup Cell Line Welcome to Agile Pulse MAX Transfectant Chamber Protocol Operator Log On to Agile Pulse MAX a setup User name admin Change hs Password Change Figure 5 1 Login Screen To enter User names and Passwords touch the Change buttons to activate a virtual keyboard User Name Enter the user s name to login Password Enter the password associated with the user account Password entry is masked with e characters to provide Increased security When the User name and Password have been entered touch OK to log in IMPORTANT Errors can be generated if power is cycled off and on too quickly Wait at least five seconds after turning power off before turning power back on Caution AgilePulse MAX Large Volume Transfection System User s Manual toll free 800 272 2775 508 893 8999 e www btxonline com 18 AgilePulse MAX System Operation 2 5 1 Waveform Generator cont d 5 1 2 Welcome Screen Sample Identification Input A Welcome Screen will be presented
30. he upper limit threshold pore diameter and total pore area become too large for the cell to repair by any spontaneous or biological process The result is irreversible damage to the cell or cell lysis Another important point to consider is the generation of heat during electroporation Heat production is directly related to current intensity which is in turn dependent on the conductivity of the material through which the electric field is applied Standard saline solutions such as PBS and many tissue culture media are highly conductive and thus will generate considerable amounts of heat when used in cell electroporation Excessive heating can be detrimental to cell viability The effects of heating can be reduced by using a low conductivity medium such as BTX s Cytoporation medium to resuspend cells prior to electroporation see also Section 3 4 for other important issues related to conductivity Although electroporation is an effective method for introducing macromolecules into cells the biological mechanisms by which cells become electroporated are not completely understood Therefore the development of specific protocols for particular applications is usually achieved by empirical adjustment of pulse parameters i e amplitude duration pulse number and interpulse interval AgilePulse MAX Tutorial AgilePulse MAX Large Volume Transfection System toll free 800 272 2775 508 893 8999 e www btxonline com Oo OO O
31. induce permeability in a larger surface area of the cell This is because of the dependence upon the cosine shown in the formula above Thus high electric fields are used in first pulses to permeabilize a large area of the cell membrane The permeabilized area on each end of a cell will be asymmetrical It will be slightly larger on the anode side than the cathode side This is because the cell has a slight natural negative charge across its membrane Golzio 2002 3 3 2 Movement of DNA or RNA Across Cell Membranes The principle force influencing movement of polynucleotides across a cell membrane during electroporation is electrophoresis Klenchin 1991 This has been shown both in vitro Andreason 1989 and in vivo Satkauskas 2002 Electrophoresis is the movement of charged particles in a solution in response to an electric field Movement of particles by electrophoresis is proportional to the total applied charge which is in turn proportional to the magnitude of the electric field times the duration of the electric field Assuming other factors such as solution viscosity are constant the movement is linearly related AgilePulse MAX Tutorial 3 3 PulseAgile Electroporation cont d AgilePulse MAX Large Volume Transfection System toll free 800 272 2775 508 893 8999 e www btxonline com 9 to the total charge This means that total distance traveled is approximately the same when half of a given electric fi
32. level of hierarchy can be saved to a USB Key inserted in the front panel by pressing the SAVE button The hierarchy structure will be maintained and copied to the USB Key The DELETE button is also available to remove the hierarchy from internal memory This deletion is permanent and the lost files cannot be recovered AgilePulse MAX System Operation Delete A Caution AgilePulse MAX Large Volume Transfection System toll free 800 272 2775 508 893 8999 www btxonline com 22 Protocol me Figure 5 7 Log File Maintenance Screen The Saving and Deleting processes are slow and dependent on the number of files being saved or deleted Do not use USB keys that will attempt to upload software onto the computer drive Keys with lt 1 GB memory are recommended e User s Manual AgilePulse MAX System Operation 5 1 Waveform Generator cont d 5 1 7 Electroporation Protocol Delivery Logs All collected data from the delivery is automatically stored on the internal Flash Memory and an inserted USB key under a unique chronological directory structure Included items are an XML file containing raw detailed system and runtime data a text log with basic runtime data and a comma separated CSV file containing pulse monitor data The directory structure is constructed in a AgilePulse MAX Log lt Year gt lt Month gt lt Day gt lt Time gt hierarchy format A maximum of 20 000 delivery
33. logs can be stored on the internal system memory Raw Data DV lt Date gt lt Time gt xml Text Log DV lt Date gt lt Time gt log CSV Data DV lt Date gt lt Time gt csv Start and Stop Date L Start and Stop Time Field 1 Field 2 Field 3 Process Completion Type Set Pulse Parameters Lj e a 2 2 2 2 Average monitor pulse voltage Average monitor pulse current Average monitor pulse resistance L j 2 2 2 2 2 2 2 All monitor pulse voltage samples All monitor pulse current samples Monitor width of each pulse Monitor interval of each pulse 2 2J aj lt Check Reading System Serial Numbers System Software Versions ee 2 A a aa Se AgilePulse MAX Large Volume Transfection System User s Manual toll free 800 272 2775 508 893 8999 e www btxonline com 23 5 1 Waveform Generator cont d 5 1 7 Electroporation Protocol Delivery Logs cont d 5 1 7 1 Text Log The text log contains the same information that appears on the delivery process screen after completion Text log data is summarized in an easy to read format and is designed to provide quick feedback regarding the delivery completion An example Log printout is presented in Figure 5 8 Date Start 2 12 2007 2 16 22 PM System Serial Number 07 0120CS Cell Line CHO Transfectant Plasmid A Chamber
34. lse MAX system 5 1 Waveform Generator The Waveform Generator is controlled by a touch screen computer It is recommended that the user touch the screen with the supplied stylus Field entries can be made with the on screen virtual keyboard Alternatively a computer keyboard may be attached via one of the USB ports The process flow is e Login e User Data Entry e Initialize Pulse Sequence e Pulse Delivery e End Process 5 1 1 Login Screen User Identification Input After power Is turned on a Login Screen is presented that requires the user to enter a user name and password combination The Login system is designed to prevent unauthorized users from accessing the system and confine authorized users to permissible activities Two types of users are defined system administrator and standard user There is only one system administrator of the system who has the ability to add remove pulse waveform protocols add remove standard user accounts and assign pulse waveform protocol access privileges to standard users The administrator always operates under the admin account Standard users are intended to be everyday users of the system who only have the ability to run pre defined pulse waveform protocols from an access list as determined by the administrator The administrator may grant a standard user the ability to modify an already defined protocol but not to add remove protocols AgilePulse MAX is factory confi
35. me where S is in S cm if in mS cm then s 103 if in uS cm then s 106 gap isin cm volume isin mL T AgilePulse MAX Large Volume Transfection System User s Manual ae toll free 800 272 2775 508 893 8999 www btxonline com 10 AgilePulse MAX Tutorial 3 4 Importance of Medium Conductivity in Electroporation cont d The table below provides some insight into how conductivity and volume affect the resistance in an electroporation chamber Examples are given for both a standard 4 mm cuvette with a maximum volume of 800 uL and a 20 mL chamber that contains either 5 mL or 20 mL volumes Medium Conductivity s Gap Volume Resistance R Pulse Current mS cm cm mL ohms 1000V Amps PBS 16 6 0 4 0 8 12 83 T 4 3 4 0 4 0 8 59 17 T 0 08 0 4 0 8 2500 0 5 PBS 16 6 0 4 1 9 519 T 4 3 4 0 4 5 9 4 106 T 0 08 0 4 5 400 3 T 0 08 0 6 6 750 1 3 PBS 16 6 0 6 6 4 277 T 4 3 4 0 6 6 17 6 57 T 0 08 0 6 2250 0 4 PBS 16 6 0 6 11 92 T 4 3 4 0 6 52 9 19 For practical reasons as the bulk of volume to be electroporated increases the conductivity must be decreased Otherwise current requirements will rapidly exceed available capacity AgilePulse MAX Large Volume Transfection System User s Manual toll free 800 272 2775 508 893 8999 e www btxonline com AgilePulse MAX
36. nce If the power is properly applied and the touch screen is illuminated then the following display is visible Cell Line Welcome to Agile Pulse MAX Transfectant Chamber Protocol Vie f Change N Change Operator Log On to Agile Pulse MAX User name admin Password OK AGE l a T j k ai L m Se ae Figure 4 5 Start Up Screen Display User s Manual AgilePulse MAX System Components amp Set Up 4 3 Setting Up the System cont d 4 3 2 Touch Screen Cleaning If the surface of the touch screen display needs to be cleaned use a standard non ammonia glass cleaner or mild detergent with warm water and a soft lint free paper or cloth towel Do not apply the cleaning solution directly to the screen to avoid liquid running into other parts of the cabinet Put a small amount of cleaner on the towel and gently rub the screen Avoid hard rubbing abrasives or harsh solvents like alcohol or ammonia This completes the set up and testing of the AgilePulse MAX System If there is a problem contact Technical Support for assistance Phone 508 893 8999 or Toll Free 800 272 2775 www btxonline com AgilePulse MAX Large Volume Transfection System toll free 800 272 2775 508 893 8999 e www btxonline com User s Manual This chapter gives a detailed description of the operation of the AgilePu
37. nel The following functions are on the front panel the Mains Line power switch illuminated when on the Emergency Stop button switch two USB ports the electrode connector and the User Touch Screen Figure 4 2 One USB port is used for a Memory Key The data logs that result from a transfection run are automatically stored on the key if the key is inserted The Emergency Stop button switch is used to stop system operation in an emergency situation This switch immediately stops all pulsing and turns off the high voltage power supply Figure 4 2 Waveform Generator Front Panel an emergency situation while pulsing is in progress Caution The Mains Line power switch should never be turned off in AgilePulse MAX Large Volume Transfection System toll free 800 272 2775 508 893 8999 e www btxonline com 4 2 2 Back Panel The back panel has two connectors an IEC Power Entry module and an Ethernet connection The Ethernet connection Is currently not functional but is built in for expansion of future versions 4 2 3 Large Volume Chamber amp Base The Large Volume Chamber consists of two flat and parallel plates with 6 mm spacing There are two luer connectors the lower luer port for connecting a syringe to load cell suspension sample into chamber This luer port also has a stopcock valve to allow for opening and closing of the port during loading and removal of sample The second luer connector locat
38. nly be used as described in this User Manual the user is required to read the User Manual and undergo training before use Certain concepts and processes are covered by patents patents pending know how and trade secrets Do not under any circumstances turn the power switch off while the system is pulsing if an emergency occurs push the emergency button on the front panel Do not connect or disconnect the Chamber electrode from the waveform generator when the pulse waveforms are being applied Do not put your fingers or any other object on the Chamber while the waveforms are being applied This system produces voltages as high as 1200 volts and may cause injury or death if used improperly The system shall not be used in the presence of flammable gas The system is not for use on humans BTX Harvard Apparatus welcomes any comments from users to improve this manual ie E AgilePulse MAX Large Volume Transfection System User s Manual toll free 800 272 2775 508 893 8999 e www btxonline com 6 AgilePulse MAX Tutorial 3 1 Applications The AgilePulse MAX system is used to deliver material into cells using pulsed electric fields It is a large capacity system capable of treating up to 20 mL of cell suspension The AgilePulse MAX system was specifically designed for the transfection of cells with polynucleotides However a variety of charged material can potentially be delivered into cells Membrane Befo
39. or filling and emptying of the chamber The Large Volume Electroporation Chambers may be re used if properly cleaned after each use Chambers can be sanitized by soaking in 1M NaOH 4 solution for 10 minutes then rinsing liberally with sterile water Chambers are not autoclave safe Alternative cleaning procedures must be validated prior to use o AgilePulse MAX Large Volume Transfection System User s Manual ae toll free 800 272 2775 508 893 8999 e www btxonline com 27 AgilePulse MAX System Operation 5 2 Transfection Process cont d 5 2 4 Electroporation Place the Chamber in the Chamber Holder and apply the selected waveform protocol It is recommended that electroporation protocols be optimized on a small scale prior to transfection in large volumes However additional optimization is often required during the scale up process The following table is provided as a starting point for protocol optimization These protocols were established by BTX Harvard Apparatus or its collaborators by optimization in cuvettes Additional information concerning transfection of specific cell tyoes may be found on the BTX Harvard Apparatus web site www btxonline com Pulse Group 1 Pulse Group 2 Cell Line Polynuc Conc ug ml Amplitude V Duration ms Pulse No Amplitude V Duration ms Pulse No cm cm CHO K1 Plasmid 50 2000 0 4 2 1000 0 8 2 HEK293 Plasmid 50 1600 0 2 2 800 0 8 4 3T3 L1 Plasmid 50 2
40. ransfection AgilePulse MAX System Components amp Set Up AgilePulse MAX Large Volume Transfection System toll free 800 272 2775 508 893 8999 e www btxonline com 16 4 3 Setting Up the System The system set up and testing procedure is as follows 1 Unpack the contents of the shipping box 2 Check for obvious signs of exterior damage If damage is noted contact BTX Technical Support before proceeding 3 Place the Waveform Generator on the top of a sturdy table 4 Connect the Chamber Base cable into the connector at the bottom right of the front panel 5 Connect the mains power cord into the back panel at the bottom right 4 3 1 Initial System Test This section will describe the process to verify mains line cable has been installed properly and the computer boots up 1 Connect the power cord to the mains 2 Turn the rocker switch on the front panel to the on position I What should happen The rocker switch Power should illuminate and the Opening Touch Screen should appear within 10 seconds If the Waveform Generator power switch fails to illuminate then return it to the OFF position O Verify that the power cord Is properly plugged into the wall It may be necessary to check the fuse on the back panel of the Waveform Generator If it is faulty replace it with the exact type fuse 240V 5A slo blo 5 x 20 mm If a problem persists call BTX Technical Support for assista
41. re Field Applied 3 2 DNA RNA Delivery into Cells Using Electroporation 3 2 1 General Electroporation Discussion Electroporation uses pulsed electric fields to create microscope pathways pores in bio membranes Their presence allows macromolecules ions and water to pass from one side of the membrane to the other As the right side bar shows when the electric field is applied the ions inside and outside the cell membrane migrate As the charge builds up on either side of the membrane pores form that permit material outside of the cell to enter If the electric field is promptly removed the pores close and the membrane reseals If the electric field duration is too long the pores increase in size and number and the cell is killed Electroporation was first used to introduce small molecules into cells in the early 1980 s and quickly became a popular research method By the late 1980 s electroporation applications were being developed for multi cellular tissues In the early 1990 s Lluis Mir of the Institute Gustave Roussy was the first to use electroporation in a human trial to treat external tumors Efficient electroporation depends on proper selection of electric field waveforms Pores begin to form in about a microsecond and reseal in seconds to minutes Therefore duration of the applied electric fields are typically in the range of tens of microseconds to tens of milliseconds Induction of electropores is affected by a number of
42. screen on the front panel The computer has a power connection on the back panel and a power switch emergency stop switch electrode connector and two USB ports on the front panel All User inputs and outputs are via the touch screen The system is controlled by a microcontroller The microcontroller accepts user inputs from the touch screen and produces the pulse waveforms A second microcontroller is used as an independent audit of the waveforms produced If the independent audit function detects a deviation from the pulse protocol the system immediately terminates pulsing The system also digitizes the output of the pulse voltage and pulse current monitors and calculates the resistance of the volume processed This is a very significant quality control function that indicates the electric fields are being applied correctly After the cells in solution are transfected a complete log of all parameters is saved in internal memory If a USB memory key is inserted the data Is also written to the key The key may also be inserted at a later time and a particular run can be saved to the key AgilePulse MAX Large Volume Transfection System toll free 800 272 2775 508 893 8999 e www btxonline com AgilePulse MAX System Components amp Set Up Figure 4 1 Waveform Generator User s Manual 14 B AgilePulse MAX System Components amp Set Up 4 2 The AgilePulse MAX Waveform Generator cont d 4 2 1 Front Pa
43. toporation Medium Properties In addition to low conductivity Cytoporation Media have other properties that contribute to successful electroporation such as physiological pH and normal osmolarity The pH is moderately buffered but addition of any reagents that could affect pH should be avoided The osmolarity of Cytoporation Media is compatible for animal cells Addition of small molecule reagents ionic or non ionic if added in substantial amounts to the medium may adversely affect osmolarity Cytoporation Media contain proprietary ingredients designed to improve the viability of cells during electroporation Cytoporation Media are RNase and DNase free Every batch is tested and certified free of the presence of both enzymes Cytoporation Media are low endotoxin media Endotoxin will adversely affect transfection of cells using electroporation Therefore a low endotoxin medium is essential The AgilePulse MAX system was designed to be used with the Cytoporation Media offered by BTX Harvard Apparatus The use of other buffers or media may invalidate the warranty o AgilePulse MAX Large Volume Transfection System User s Manual ae toll free 800 272 2775 508 893 8999 e www btxonline com AgilePulse MAX Tutorial 3 6 References Andreason GL Evans GA Optimization of electroporation for transfection of mammalian cells 1989 Anal Biochem 180 269 219 Bartoletti D C Harrison G 1 amp Weaver
44. which they are not intended or designed or if they are used by untrained or unqualified personnel The user must read this manual carefully before the instrument is placed into operation Removing the cover may void the warranty Do not connect or disconnect the high voltage cable with the high voltage enabled To connect or disconnect the cable turn line power off and unplug line mains cord Do not touch the electrode tip while the waveforms are being applied If a problem occurs during a run push the STOP RESET button on the front panel If there are any questions about the operation of this instrument call BTX Customer service at 1 800 272 2775 or 1 508 893 8999 ie E AgilePulse MAX Large Volume Transfection System User s Manual toll free 800 272 2775 508 893 8999 e www btxonline com 4 amp B Introduction The AgilePulse MAX system was developed to transfect large volumes of cells for commercial applications using pulsed electric fields electroporation The system has been designed for use in commercial processing The system has four components the Waveform Generator Subsystem the ex vivo large volume Chamber Electrode Subsystem low conductivity Cytoporation medium and User Manual These components were all designed to operate as an integrated system Using other components may damage the system and will certainly provide degraded performance and invalidate void the warranty Note The AgilePulse MAX
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